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I IDENTIFICATION of PROTEOLYTIC CLEAVAGE and ARGININE MODIFICATIONS by CHEMICAL LABELING and MASS SPECTROMETRY by MAHESHIKA S.K

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I IDENTIFICATION of PROTEOLYTIC CLEAVAGE and ARGININE MODIFICATIONS by CHEMICAL LABELING and MASS SPECTROMETRY by MAHESHIKA S.K IDENTIFICATION OF PROTEOLYTIC CLEAVAGE AND ARGININE MODIFICATIONS BY CHEMICAL LABELING AND MASS SPECTROMETRY by MAHESHIKA S.K. WANIGASEKARA Presented to the Faculty of the Graduate School of The University of Texas at Arlington in Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY THE UNIVERSITY OF TEXAS AT ARLINGTON December 2017 i This dissertation is dedicated to my mother and late father who left us on April 02nd 2001, my husband Dhanuka and my son Mayon for their love and support. ii Acknowledgements First and foremost, I owe sincere and earnest thankfulness to my research advisor, Dr. Saiful Chowdhury. My sincere gratitude and profound thanks goes to you for welcoming me into your research group. I am also grateful for your patient guidance and supervision throughout my graduate career. I express my sincere thanks to my committee members, Dr. Kevin Schug, Dr. Daniel Armstrong and Dr. Jonguyn Heo, for their valuable feedback during my UTA research career. I would like to express my deepest appreciation to the Department of Chemistry and Biochemistry and all department staff members for their kind assistance from the beginning to the end of my graduate studies at the University of Texas at Arlington. I was also fortunate to have a group of wonderful friends in my research group, who helped me in many ways during my time at UTA. I am eternally thankful for my husband for his endless love, and support. None of this could be possible without him. Words cannot express how grateful I am for everything he has done for me. He sacrificed immensely for me so I could obtain my Ph.D. I am very grateful to my brother, Eranda Wanigasekara, who has helped me accomplish many things in my life. Finally, a special thanks to my sister-in-law, Tharanga, and my husband’s family for their enormous support and encouragement throughout the years, and especially during my graduate studies. iii Abstract IDENTIFICATION OF PROTEOLYTIC CLEAVAGE AND ARGININE MODIFICATIONS BY CHEMICAL LABELING AND MASS SPECTROMETRY Maheshika S.K. Wanigasekara, PhD The University of Texas at Arlington, 2017 Supervising Professor: Dr. Saiful M. Chowdhury Post-translational modification of proteins plays a significant role in the regulation of cellular processes. During the translation, process proteins undergo different modifications, which are known as post-translational modifications (PTMs). Due to their low abundances, PTM analysis presents several challenges; therefore, efficient and sensitive PTM detection methods are required. Arginine is an essential amino acid in a cell, which undergoes several kinds of PTMs. Finding these functional arginine residues in a protein is a challenging task. The task was successfully addressed, and the findings are not only reported herein, but an assessment was recently reported covering the new chemical labeling methods developed for identifying functional arginine residues of proteins by comparing two widely used arginine labeling reagents—1,2-cyclohexanedione (CHD) and phenylglyoxal (PG). This dissertation combined the author’s and her research team’s previous studies with bio-orthogonal chemistry and quantitative mass spectrometry-based proteomics to develop a new approach for the selective enrichment of reactive arginine residues in proteins in complex samples. The primary achievement was the development of a novel arginine-specific, azide-tagged CHD analog, which enables labeling of reactive arginine residues for further studies. For large-scale samples, the workflow from this labeling process is adaptable for gel-based pre-separation. This research established a promising strategy for the effective profiling of reactive arginine residues in large- scale studies. N-terminal modifications of proteins can interpret the functions and stability of proteins, affecting their expression, activation, or degradation. Moreover, selective enrichment iv of N-terminal peptides from a complex mixture is a difficult challenge in the proteomics field. Currently we are working with two novel reagents, which have innovative properties to enrich the N-terminal peptides and to generate marker ions from the N-term-labeled peptides during tandem mass spectrometry. These studies will significantly contribute to proteomics and to further research in the bio-analytical mass spectrometry field. v Table of Contents Acknowledgements…………………………………………………………...………………………….iii Abstract………………………………………………………………………………………………...….iv Lis of Illustrations…………………………………………………………………………………………ix List of Tables……………………………..……………………………………………..…………….....xv List of Abbreviations………………..……..……………………………………………………………xvi Chapter 1 Introduction………………………………………………………………………….……………...……1 1.1 Thesis Organization………………………………………………………………………………….1 1.2 Post-translational modifications……………………………………………………………...……..1 1.3 Proteins ………………………...……………………………………………….…………………….3 1.4 Arginine.……….…………………………………………………………..……………………….…4 1.5 Arginine Post-translational modifications……….…………………………………………….…...5 1.6 Proteolytic Cleavage………………………………………..…..…………………………………...6 1.7 Mass Spectrometry…………………..…………...…………………………………………….…...7 1.8 Soft Ionization Methods..………….…………………………………………………………….…..8 1.9 Ion Activation………………………………………………..……………………….……………...10 1.10 Mass Analyzers……………………………………...…………………………………………….11 1.11 Detectors……..……………………...…………………………………………………………….12 Chapter 2 Evaluation of chemical labeling methods for identifying functional arginine residues of proteins by mass spectrometry…………………………….....……………………..13 2.1 Abstract………...…………………………………………………………………………..………..13 2.2 Introduction..……………………… ……………………………………………………………..…14 2.3 Materials and Methods……………………………………………..……………………………...17 2.3.1 Chemicals and Materials.……...………………………………………………………...…..….17 vi 2.3.2 Protein digestion………………………………………………………………………................17 2.3.3 Labeling of arginine residues in peptides……………………………………………………...18 2.3.4 Chemical modification of intact bovine serum albumin (BSA) and lysozyme…………..….18 2.4 Results and Discussion…..……………………………………………………………….............20 2.4.1 Modification by CHD …………………...………………………………………………………..20 2.4.2 Arginine modification of peptides by phenylglyoxal (PG)…………………………………….24 2.4.3 Arginine modification of intact bovine serum albumin (BSA) by CHD…………..………….28 2.4.4 Arginine modification in lysozyme…………………………..…………………………..……...32 2.4.5 Structural analysis of reactive arginine residues………………………...………...………….29 2.5 Conclusions………..………………………………………………………………………………..32 2.6 Acknowledgements…………………………..…………………………………………………….34 2.8 Supporting information………………..…………………………………………………………...35 Chapter 3 Enrichment and identification of reactive arginine residues in proteins by bio- orthogonal click chemistry and mass spectrometry……………………………………...…….45 3.1 Abstract……..………………………………………….……………………………………………45 3.2 Introduction………..………………………………………………………………………..............46 3.3 Experimental section……………..………………………………………………………………...48 3.3.1 Materials………………………………………………………………………..…………….…...48 3.3.2 Sample preparation …………………………………..……………………………….………...48 3.3.2.1 Chemical modification of arginine residues in peptides………………………..…...……..49 3.3.2.2 Chemical modification of arginine residues in proteins………………………...…………..50 3.3.2.3 The feasibility of click chemistry-based peptide enrichment………………………………50 3.3.2.4 Instrument Set up …………………………..…………………………………………..……..51 3.4 Results and Discussion………………………………………………………………………........51 vii 3.4.1 Modification of Peptides by CHD-Azide reagent………………………………………………52 3.4.2 Tandem mass spectrometry studies of the peptides ………………………………….……..53 3.4.3 The feasibility of click chemistry-based peptide enrichment……………..………………….54 3.4.4 Modification of RNase A Protein by CHD-Azide compound.………...………………………54 3.5 Conclusions…………………………...………………………………………………………..…...56 3.6 Acknowledgements…………………………………………..…………………………………….57 Chapter 4 A novel mass spectrometry cleavable reagent that enables identification of proteolytic cleavage …………….....……………………………………………………………...….66 4.1 Abstract…………………………...……………..…………………………………………………..66 4.2 Introduction…………….…..………………………………………………………………………..69 4.3 Materials and methods …………………………..………………………………………………..70 4.3.1 Spiking Studies ……………………..……………………………………………………………70 4.4 Results and Discussion…………………..………………………………………………………..74 4.5 Conclusion……………...……………………………………………………………………….......75 Chapter 5 Summary and Future Work…………………………..…………………………………76 References…..………………………………………………………………………………………….79 Appendix 1 Copyright Clearance…………………………………………………..……………….87 Vita……………………….…..…………………………………………………………………………...88 viii List of Illustrations Figure 1-1 Common Post-Translational Modifications ………………………………..….….………2 Figure 1-2 Basic structure of an amino acid……………………………...……………………………3 Figure 1-3 Structure of arginine amino acid…………………………..……………………………….5 Figure 1-4 – Schematic diagram of positional proteomics workflow…………………..……………6 Figure 1-5 Schematic of the main components of a mass spectrometer……………..……………7 Figure 1-6 Matrix-assisted laser desorption ionization (MALDI) in mass spectrometry……...…...9 Figure 1-7 Electrospray ionization method in mass spectrometry……………………...………....11 Figure 1-8 Interpretation of b and y ion formation in peptides……………………...………………11 Figure 2-1 Graphical abstract……………………...…………………………………………………..14 Fig. 2-2 The structures of the modifications resulting in the label peptides with 1,2- cyclohexanedione and phenylglyoxal. I-1,2-cyclohexanedione, II-phenylglyoxal, Ia-Arginine- CHD covalent product, IIa-partial modified arginine-PG
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